Cytotoxic Metabolites from Calophyllum tacamahaca Willd.: Isolation and Detection through Feature-Based Molecular Networking

Isocaloteysmannic acid (1), a new chromanone, was isolated from the leaf extract of the medicinal species Calophyllum tacamahaca Willd. along with 13 known metabolites belonging to the families of biflavonoids (2), xanthones (3–5, 10), coumarins (6–8) and triterpenes (9, 11–14). The structure of the new compound was characterized based on nuclear magnetic resonance (NMR), high-resolution electrospray mass spectrometry (HRESIMS), ultraviolet (UV) and infrared (IR) data. Its absolute configuration was assigned through electronic circular dichroism (ECD) measurements. Compound (1) showed a moderate cytotoxicity against HepG2 and HT29 cell lines, with IC50 values of 19.65 and 25.68 µg/mL, respectively, according to the Red Dye method. Compounds 7, 8 and 10–13 exhibited a potent cytotoxic activity, with IC50 values ranging from 2.44 to 15.38 µg/mL, against one or both cell lines. A feature-based molecular networking (FBMN) approach led to the detection of a large amount of xanthones in the leaves extract, and particularly analogues of the cytotoxic isolated xanthone pyranojacareubin (10).


Introduction
The genus Calophyllum (Calophyllaceae) includes approximately 200 species, distributed across all tropical regions. They are traditionally used against many ailments, including ulcers, malaria, tumor, infections, eye diseases, pain, inflammation and rheumatism [1,2]. This genus is an important source of bioactive natural products, including coumarins, xanthones, chromanones and triterpenes [3,4]. Xanthones and coumarins from Calophyllum species are known to possess cytotoxic, antiviral, antimicrobial, antiparasite, analgesic, anti-inflammatory and chemopreventive properties [5,6]. (+)-Calanolide A, a ium of the University of La Réunion for confirmation of identification, with the following accession number: REU024075.

Extraction and Isolation
Leaves of C. tacamahaca were dried at 40 • C for 48 h and powdered. An accelerated solvent extractor (ASE 300 Dionex) was used to exhaustively extract the ground material (237.0 g). Four successive extractions were performed at 40 • C with EtOAc. The extract was evaporated under reduced pressure at 38 • C to obtain 20.3 g of crude extract.

Conformational Study for UV-ECD Calculations
Conformational analysis was performed by stochastic exploration of the potential energy surface (PES) using the simulated annealing algorithm proposed by the Ampac11 software and combined with semi-empirical levels RM1 [15]. For the annealing, a geometry optimized at GD3BJ-B3LYP/6-311G(d,p) level was used as a starting structure. The GD3BJ term stands for empirical dispersion which was added with the D3 version of Grimme's dispersion with Becke-Johnson damping (GD3BJ) [16]. During each annealing, only the dihedral angles of this initial geometry were allowed to relax, the bond lengths and the valence angles were kept constant. A set of 24 geometries (the conformations with energy lower than 3 kcal mol −1 compared to the lower energy conformation) were selected for each diastereomer from the structures generated by 4 simulated annealing algorithms, each performed either with an initial geometry with some dihedral angles modified or with a different annealing temperature. Then, these geometries were fully optimized (i.e., all internal coordinates released) using GD3BJ-B3LYP/6-311G(d,p) level.

Calculation of Averaged UV and ECD Spectra
Based on the GD3BJ-B3LYP/6-311G(d,p) optimized geometries, the UV and ECD spectra were calculated using time-dependent density functional theory (TDSCF-DFT) with CAM-B3LYP functional and 6-31++G(d,p) basis set and with the SMD(CH 3 OH) solvation model. SMD indicates the implicit solvent model used which is a dielectric continuum model that simulates the average effects of the solvent [17]. Calculations were performed for vertical 1A singlet excitation for 50 states. For a comparison between theoretical results and the experimental values, the calculated UV and ECD spectra have been modeled with a gaussian function using a half-width of 0.33 eV. Due to the approximations of the theoretical model used, an almost constant offset was observed between measured and calculated wavenumbers. Using UV spectra, all frequencies were calibrated by a factor of 1.05. Gaussian 16 package [18] was used to perform all calculations. It should be noted that similar calculations were performed using the LC-whPBE functional instead of CAM-B3LYP (SMD(CH 3 OH)/LC-whPBE/6-31++G(d,p)//GD3BJ-B3LYP/6-311G(d,p)) and led to a similar result, which is not presented here.

In Vitro Cytotoxic Assay
HepG2 (human liver cancer) and HT29 (human colon and colorectal adenocarcinoma) cell lines were used to assess the toxicity of samples. In the performed assay, cytotoxicity was expressed as a concentration-dependent reduction in the uptake of the vital dye Neutral Red (NR) when measured 24 h after treatment. NR is a weak cationic dye that readily penetrates cell membranes by non-diffusion and accumulates intracellularly in lysosomes. Alterations of the sensitive lysosomal membrane lead to lysosomal fragility and other changes that gradually become irreversible. This results in a decreased uptake and binding of NR in non-viable cells. HT29 (ATCC ® HTB-38™) and HepG2 (ATCC ® HB-8065™), low passage number (<50), were cultivated into DMEM (Dulbecco's Minimum Essential Medium, PAN BIOTECH. lot 1874561) supplemented with penicillin 100 IU/mL and streptomycin 100 µg/mL (PAN BIOTECH, Lot 945514), and 10% of inactivated calf serum (PAN BIOTECH, Lot P56314), pH 7.2, freshly prepared, stored no longer than 1 week. Cells were seeded into 96-well tissue culture plates (0.1 mL per well) at a concentration of 1.10 5 cells/mL and incubated at 37 • C (5% CO 2 ) until semi-confluent. The test material was diluted into sterile DMSO (stock solutions 0.1, 1 and 10 mg/mL) at final concentrations ranging from 0.1 to 250 µg/mL. The culture medium was decanted and replaced by 100 µL of fresh medium containing the various concentrations of the test material; then, cells were incubated for 24 h at 37 • C (5% CO 2 ). At the end of the incubation period, cells were placed into Neutral Red medium (50 µg/mL NR in complete medium) and incubated for 3 h at 37 • C, 5% CO 2 . Then, the medium was removed, and cells were washed three times with 0.2 mL of HBSS to remove excessive dye. The Neutral Red medium was removed and the distaining solution (50% ethanol, 1% acetic acid, 49% distilled water; 50 µL per well) was added into the wells. Then, the plates were shaken for 15-20 min at room temperature in the dark. The test samples and controls were run in triplicates in three independent experiments. A fluorescence-luminescence reader Infinite M200 Pro (TECAN) was used to measure the degree of membrane damage (i.e., the increase in released NR). For each well, the Optical Density (OD) was read at 540 nm. The results obtained for test material wells were compared to those of untreated control wells (HBSS, 100% viability) and converted to percentage values. The concentrations of the test material causing a 50% release of the preloaded NR (IC 50 ) compared to the control culture were calculated using software Phototox Version 2.0. The mean OD value of blank wells (only NR desorbed solution) was subtracted from the mean OD value of three test/untreated wells.

Feature-Based Molecular Networking
The leaf crude extract of C. tacamahaca as well as the isolated metabolites were profiled by UHPLC-QqTOF-MS/MS in a mass range from m/z 50 to 1200 using positive (+) mode for the ESI source. The following parameters were used: end plate offset at 500 V; nebulizer gas pressure at 3.5 bar; dry gas flow at 12 L/min; drying temperature at 200 • C; acquisition rate at 4.0 Hz. The capillary voltage was set at 4500 V, with a fragmentation energy of 20-40 eV. The UHPLC conditions were as follows: sample concentrations: 5 mg/mL (crude extract), 0.2 mg/mL (isolated compounds) in 100% MeOH, injection volume: 2 µL, column temperature: 40 • C, elution gradient of H 2 O-CH 3 CN with 0.1% HCO 2 H (98:02 over 2 min, 98:02 to 0:100 over 12 min, 0:100 over 3 min) at a flow rate of 0.5 mL/min. Raw data obtained from the crude extract analysis were converted into open format .mzXML using software Bruker Compass DataAnalysis Version 4.2 and processed using software MZmine Version 2.53 [19][20][21]. Then, a feature-based molecular network (FBMN) was created on the GNPS platform [22], and it is available via the following link https://gnps.ucsd.edu/ ProteoSAFe/status.jsp?task=f0c193d2141d463ba34af46df7bfe57c (accessed on 29 March 2022). The Mzmine MS/MS data processing comprises .mzXML file import, MS peak detection, ADAP chromatogram builder, chromatogram deconvolution, isotopic peaks grouper, alignment, filtering, fragment search, adduct search and spectra normalization. Setting parameters were as follows: positive ionization mode, centroid detection, MS1 peak detection limit: 1 E 3, MS2 peak detection limit: 1 E 2, m/z tolerance: 10 ppm, peak/top edge ratio: 2, peak duration range: 0.03-1 min, m/z range for MS2 pairing: 0.02 Da, RT range for MS2 pairing: 0.1 min, representative isotope: most intense, alignment weight for m/z: 75, weight for RT: 25, filtering RT tolerance: 0.1 min, filtering m/z tolerance: 0.001 m/z, adduct search [M+Na] + , [M+NH 4 ] + , spectra normalization type: average intensity. Processed files including an mgf and a csv file were uploaded to the GNPS platform. An FBMN was then developed using the Advanced Analysis Tools-Feature Networking workflow [23]. Advanced Network Options parameters were as follows: min pair cos: 0.7, minimum matched fragment ions: 6, network topK: 10, maximum connected component size: 100, mass tolerance for precursor and fragment ions: 0.02 Da. The output was imported into Cytoscape Version 3.8.2 in order to visualize the network. Node annotations were performed manually for isolated compounds and with GNPS spectral databases (score threshold: 0.7) and In Silico MS/MS DataBase ISDB (score threshold: 0.2) [24].
been published so far and are provided here (Section 2 and Figures S11 to S15). The structure of the new compound 1 was established based on 1D and 2D NMR, IR and UV spectroscopic and HRESIMS spectrometric data.
The absolute configuration of (1) was established by ECD by comparing the measured spectra with those calculated using DFT and TD-DFT for diastereomers (4S,10R,11R) and (4R,10R,11R) according to the previous NMR analysis (Figure 3). The absolute configuration of (1) was established by ECD by comparing the measured spectra with those calculated using DFT and TD-DFT for diastereomers (4S,10R,11R) and (4R,10R,11R) according to the previous NMR analysis (Figure 3).
The UV and ECD spectra of (4S,10R,11R) and (4R,10R,11R) were built, respectively, from the individual spectra of the A1-6 and B1-6 conformations weighed by their Boltzmann population (Appendix A). The comparison of the calculated UV spectra for the two diastereomers showed a good agreement with the measured spectrum, without allowing to establish the absolute configuration of the C-4 atom. Furthermore, the calculated ECD spectra showed a clear sign difference around 215 nm: positive bands for (4S,10R,11R) and negative bands for (4R,10R,11R) ( Figure 5A-D). Comparison with the corresponding measured spectrum showed excellent agreement with that calculated for the (4S,10R,11R) configuration ( Figure 5A-D). In particular, The UV and ECD spectra of (4S,10R,11R) and (4R,10R,11R) were built, respectively, from the individual spectra of the A 1-6 and B 1-6 conformations weighed by their Boltzmann population (Appendix A). The comparison of the calculated UV spectra for the two diastereomers showed a good agreement with the measured spectrum, without allowing to establish the absolute configuration of the C-4 atom. Furthermore, the calculated ECD spectra showed a clear sign difference around 215 nm: positive bands for (4S,10R,11R) and negative bands for (4R,10R,11R) ( Figure 5A-D). The absolute configuration of (1) was established by ECD by comparing the measured spectra with those calculated using DFT and TD-DFT for diastereomers (4S,10R,11R) and (4R,10R,11R) according to the previous NMR analysis (Figure 3).
The UV and ECD spectra of (4S,10R,11R) and (4R,10R,11R) were built, respectively, from the individual spectra of the A1-6 and B1-6 conformations weighed by their Boltzmann population (Appendix A). The comparison of the calculated UV spectra for the two diastereomers showed a good agreement with the measured spectrum, without allowing to establish the absolute configuration of the C-4 atom. Furthermore, the calculated ECD spectra showed a clear sign difference around 215 nm: positive bands for (4S,10R,11R) and negative bands for (4R,10R,11R) ( Figure 5A-D). Comparison with the corresponding measured spectrum showed excellent agreement with that calculated for the (4S,10R,11R) configuration ( Figure 5A-D). In particular, Comparison with the corresponding measured spectrum showed excellent agreement with that calculated for the (4S,10R,11R) configuration ( Figure 5A-D). In particular, the band around 215 nm is positive as in the measured spectrum. This ECD analysis therefore confirmed the R-configuration of the C-10 and C-11 atoms, but also unambiguously established that the C-4 atom is of absolute configuration S. Consequently, compound 1 has the absolute configuration (4S,10R,11R).

Cytotoxic Activity of the Isolated Compounds
Ten isolated compounds were evaluated for their cytotoxic properties against the two cancer cell lines HepG2 and HT29. Due to their paucity, compounds 3-5 and 14 were not evaluated . Compounds 7, 8, 10, 11, 12 and 13 showed a potent activity against one or both cell lines, with IC 50 values ranging from 2.44 to 15.38 µg/mL ( Table 2). The new compound 1, as well as compound 6, exhibited a moderate activity against both cell lines with IC 50 values ranging from 15.98 to 25.68 µg/mL. The triterpenes 11-13 showed a potent activity, whereas triterpene 9 exhibited only a weak activity, suggesting that the presence of the acetoxy group in 9 could decrease its cytotoxic potential.
These results also suggest that the cis-configuration of the methyl groups in C-10 and C-11 of compounds 7 and 8 leads to a higher cytotoxic activity than the trans-configuration (compounds 1 and 6).

Feature-Based Molecular Networking Analysis of the Crude Extract
A feature-based molecular networking (FBMN) [23] approach was performed in order to provide more information about the chemodiversity of the species and to detect additional cytotoxic metabolites by highlighting close analogues of the bioactive isolated compounds. For this purpose, leaf EtOAc extract was subjected to an UHPLC-HRESIMS/MS analysis and a molecular network (MN) was generated with the FBMN tool on the GNPS platform.

Chemodiversity of the Species
A molecular network (MN) comprising 520 features and 55 clusters (two features at least) was obtained ( Figure 6). Squared orange nodes correspond to the isolated  compounds 1, 3, 4, 5, 6, 8 and 10. Green nodes correspond to spectral matches on GNPS or ISDB databases. The edge thickness correlates with the cosine score (CS) value (0.7-1) between two nodes. Relatively few consistent spectral matches on GNPS or ISDB databases were obtained. Based on these matches, the largest cluster C1 (43 nodes) could correspond to xanthones. Two nodes correspond to the isolated xanthones 5 and 10, and three nodes were putatively identified as xanthones previously reported in the genus Calophyllum: 6-deoxyisojacareubin, mammea B/BA and caloxanthone. Seven nodes could correspond to xanthones reported in close botanical families of Calophyllaceae: elliptoxanthone B (Hypericaceae), garcinexanthone C (Clusiaceae), celebixanthone (Hypericaceae), nigrolineaxanthone K (Clusiaceae), garcinone A (Clusiaceae), hypericumxanthone B (Hypericaceae) and garcimangosone C (Clusiaceae).
Cluster C8 is another cluster of xanthones, containing the isolated metabolites 3 and 4, as well as one node putatively identified as caloxanthone H. The latter was previously reported in the genus Calophyllum.
The new compound 1 is located in cluster C5. In the latter, one node corresponds to a close analogue of 1 (m/z 423.1785, CS > 0.9). Based on ISDB matches, this close analogue was putatively identified as isochapelieric acid, a compound isolated from the species Calophyllum calaba [38].
These observations are consistent with the data in the literature, indicating that xanthones and chromanones are largely represented in the genus Calophyllum. Relatively few consistent spectral matches on GNPS or ISDB databases were obtained. Based on these matches, the largest cluster C1 (43 nodes) could correspond to xanthones. Two nodes correspond to the isolated xanthones 5 and 10, and three nodes were putatively identified as xanthones previously reported in the genus Calophyllum: 6-deoxyisojacareubin, mammea B/BA and caloxanthone. Seven nodes could correspond to xanthones reported in close botanical families of Calophyllaceae: elliptoxanthone B (Hypericaceae), garcinexanthone C (Clusiaceae), celebixanthone (Hypericaceae), nigrolineaxanthone K (Clusiaceae), garcinone A (Clusiaceae), hypericumxanthone B (Hypericaceae) and garcimangosone C (Clusiaceae).
Cluster C8 is another cluster of xanthones, containing the isolated metabolites 3 and 4, as well as one node putatively identified as caloxanthone H. The latter was previously reported in the genus Calophyllum.
The new compound 1 is located in cluster C5. In the latter, one node corresponds to a close analogue of 1 (m/z 423.1785, CS > 0.9). Based on ISDB matches, this close analogue was putatively identified as isochapelieric acid, a compound isolated from the species Calophyllum calaba [38].
These observations are consistent with the data in the literature, indicating that xanthones and chromanones are largely represented in the genus Calophyllum.

Detection of Additional Bioactive Metabolites
Two analogues of the cytotoxic isolated compound pyranojacareubin (10) have been detected in cluster C1 (Figure 7) at m/z 395.1475 and m/z 327.0854. Based on structureactivity relationship, these analogues could correspond to cytotoxic metabolites. They were putatively identified as muxiangrine I and elliptoxanthone B, according to ISDB matches. To the best of our knowledge, no cytotoxic properties have been reported in the literature for these compounds. As these identifications are highly hypothetical, it would be necessary to target the isolation of these two compounds, to identify them and assess their biological properties in a future work.

Detection of Additional Bioactive Metabolites
Two analogues of the cytotoxic isolated compound pyranojacareubin (10) have been detected in cluster C1 (Figure 7) at m/z 395.1475 and m/z 327.0854. Based on structureactivity relationship, these analogues could correspond to cytotoxic metabolites. They were putatively identified as muxiangrine I and elliptoxanthone B, according to ISDB matches. To the best of our knowledge, no cytotoxic properties have been reported in the literature for these compounds. As these identifications are highly hypothetical, it would be necessary to target the isolation of these two compounds, to identify them and assess their biological properties in a future work.

Conclusions
Fourteen metabolites (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) were isolated from the EtOAc leaf extract of C. tacamahaca. To the best of our knowledge, compound 1 was reported for the first time. Six compounds (7, 8, 10, 11, 12 and 13) showed a potent cytotoxicity against HepG2 and/or HT29 cell lines. The FBMN approach allowed the detection of a large amount of xanthones in the extract, including two close analogues of the cytotoxic compound 10. Xanthones are well known for their cytotoxic properties [2], so the results of this study suggest that C. tacamahaca leaves are a significant source of cytotoxic metabolites. These compounds could be interesting candidates for future therapeutic applications. Nevertheless, further studies are needed to evaluate their in vivo anticancer activity, as well as their mechanism of action, and thus confirm their therapeutic potential.

Conclusions
Fourteen metabolites (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) were isolated from the EtOAc leaf extract of C. tacamahaca. To the best of our knowledge, compound 1 was reported for the first time. Six compounds (7, 8, 10, 11, 12 and 13) showed a potent cytotoxicity against HepG2 and/or HT29 cell lines. The FBMN approach allowed the detection of a large amount of xanthones in the extract, including two close analogues of the cytotoxic compound 10. Xanthones are well known for their cytotoxic properties [2], so the results of this study suggest that C. tacamahaca leaves are a significant source of cytotoxic metabolites. These compounds could be interesting candidates for future therapeutic applications. Nevertheless, further studies are needed to evaluate their in vivo anticancer activity, as well as their mechanism of action, and thus confirm their therapeutic potential.